Development of Azoreductase-Activated Precursors for Efficient Hydropersulfide Release via 1,6-Elimination

Hydropersulfides (RSSH) are increasingly recognized for their potent redox-modulating and cytoprotective properties, yet their therapeutic potential remains underexplored due to their chemical instability. Here, we report the design and optimization of azoreductase (AzoR)-responsive RSSH donors for programmed release. We explore azo-linked precursors that undergo AzoR-mediated reduction to form phenylamino intermediates, which are designed to trigger RSSH release via spontaneous 1,6-elimination. A series of precursors was synthesized to evaluate the structure-activity relationships governing elimination efficiency. Direct attachment of aliphatic or aromatic RSSH moieties at the benzylic position of the azobenzene core (azodisulfides, <b>AzDS-1, AzDS-2, AzDS-3</b>) resulted in stable intermediates that failed to eliminate RSSH under physiological conditions. Even an electron-rich substituent such as a methoxy group on the azobenzene core was not sufficient to drive the elimination. To improve leaving group ability, we synthesized an azoperthiocarbonate donor (<b>AzPTC</b>) that enabled AzoR-triggered RSSH release but also underwent undesired nonenzymatic hydrolysis. Finally, incorporation of a hydrolytically stable perthiocarbamate yielded the precursor azoperthiocarbamate (<b>AzPTB</b>) that released RSSH selectively upon AzoR activation via 1,6-elimination, decarboxylation, and an intramolecular cyclization cascade. <b>AzPTB</b> demonstrates high enzymatic turnover and excellent stability under neutral aqueous conditions. These results provide key structural parameters that govern RSSH release via 1,6-elimination and establish <b>AzPTB</b> as a robust platform for site-selective delivery. This work expands the chemical biology toolkit for probing RSSH signaling and supports future efforts in redox-based therapeutic development.